In Vitro Methylation of theBsuRI (5′-GGCC-3′) Sites in the E2a Region of Adenovirus Type 2 DNA Does Not Affect Expression inXenopus laevisOocytes

The early region 2a (E2a) of adenovirus type 2 (Ad2) DNA codes for a 72,000-dalton DNA-binding protein and is expressed in the Ad2-transformed hamster cell line HE1 but not in cell lines HE2 and HE3 (H. Esche, J. Virol. 41 :1076-1082, 1982; K. Johansson et al., J. Virol. 27 :628-639, 1978). An inverse correlation between DNA methylation at the 5′-CCGG-3′ sites of the E2a region and of gene expression in these cell lines has been observed (L. Vardimon et al., Nucleic Acids Res. 8 :2461-2473, 1980). When the cloned E2a region of Ad2 DNA is methylated in vitro at the 5′-CCGG-3′ sites, the gene is not transcribed after being injected into the nuclei of Xenopus laevis oocytes, whereas unmethylated DNA is expressed (L. Vardimon et al., Eur. J. Cell Biol. 25 :13-15, 1981; L. Vardimon et al., Proc. Natl. Acad. Sci. U.S.A. 79 :1073-1077, 1982). These data demonstrate that DNA methylation is directly involved in the shut-off of transcription. In the present communication we investigated in detail the control region of the gene for the DNA-binding protein in Ad2-transformed cell lines and showed that the first late control region (map coordinate 72 on the viral DNA) of the E2a region is present in its entirety in cell lines HE1, HE2, and HE3. The Hae III sites (5′-GGCC-3′) in the E2a region in all three cell lines were not methylated. When the DNA methyltransferase Bsu RI was used, all 5′-GGCC-3′ sites in the cloned E2a region of Ad2 DNA were methylated in vitro. It was shown that methylation of these sites did not inhibit the expression of this viral gene in X. laevis oocytes. Thus, for methylation to affect gene expression in the E2a region it has to occur at specific sites (e.g., 5′-CCGG-3′) which may be different for other genes.